In view of the critical role of glutathione metabolism in the mediation of various cellular processes and renewed interest in the involvement of glutathione in neoplasia and carcinogenesis, characterization of metabolic intermediates and their relation to individual enzymatic steps is urgently needed. We have succeeded in separating sulfhydryl oxidase ("glutathione oxidase") from Gamma-glutamyltransferase ("Gamma-glutamyl transpeptidase") through covalent chromatography of detergent-solubilized membranes, which offers new opportunities for clarifying their respective roles in normal and diseased states. Specific aims are to establish the organ and tissue distributions of these enzymes, to elucidate their mechanism and metabolic role(s) with respect to disulfide bond generation, to delineate molecular events which occur during protein folding as catalyzed by sulfhydryl oxidase, and to examine what role, if any, the oxidase and the transferase may play in inherited or induced metabolic disorders involving disulfide bond formation, such as cystinosis, glutathionuria, tumorogenesis and cataractogenesis. Immunofluorescent histochemical studies of mammary, kidney, ocular and other tissues, together with specific enzymatic assays will be used to examine the levels of sulfhydryl oxidase and glutamyltransferase activities in normal and diseased animal models. NPLC analyses will be utilized in defining individual intermediates of glutathione metabolism by tissue preparations and by purified enzymes incubated in varied ratios. Catalytic mechanisms willl be evaluated using kinetic measurements, selected inhibitors and trapping agents. Chemical and physicochemical procedures will be used to further characterize the molecular structure of pure sulfhydryl oxidase, after solubilization in octylglucoside. The role of disulfide bond generation in processes of polypeptide chain folding to yield biologically functional protein structures will be assessed by examination of folding pathways of ribonuclease A in the presence and absence of sulfhydryl oxidase, using a combination of peptide mapping and immunochemical procedures, such studies may then be extended to other proteins. It is anticipated that long-term benefits of elucidating biochemical and histological relationships of sulfhydryl oxidase and glutamyltransferase will include greater understanding and improved treatment of metabolic disorders involving disulfide bonds.